Linagliptin treatment is associated with altered cobalamin (VitB12) homeostasis in mice and humans

Linagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor used for the treatment of type 2 diabetes, with additional beneficial effects for the kidney. Treatment of mice with linagliptin revealed increased storage of cobalamin (Cbl, Vitamin B12) in organs if a standard Cbl diet (30 µg Cbl/kg chow) is given. In order to translate these findings to humans, we determined methylmalonic acid (MMA), a surrogate marker of functional Cbl homeostasis, in human plasma and urine samples (n = 1092) from baseline and end of trial (6 months after baseline) of the previously completed MARLINA-T2D clinical trial. We found that individuals with medium Cbl levels (MMA between 50 and 270 nmol/L for plasma, 0.4 and 3.5 µmol/mmol creatinine for urine, at baseline and end of trial) exhibited higher MMA values at the end of study in placebo compared with linagliptin. Linagliptin might inhibit the N-terminal degradation of the transcobalamin receptor CD320, which is necessary for uptake of Cbl into endothelial cells. Because we demonstrate that linagliptin led to increased organ levels of Cbl in mice, sustained constant medium MMA levels in humans, and inhibited CD320 processing by DPP-4 in-vitro, we speculate that linagliptin promotes intra-cellular uptake of Cbl by prolonging half-life of CD320.


MMA ELISA.
To assess effects of linagliptin on Cbl homeostasis in humans, MMA levels were measured in urine (uMMA) and plasma (pMMA) samples from the MARLINA-T2D clinical trial. The rational to measure these surrogate markers of Cbl homeostasis is that pMMA and uMMA levels are increased if the vitamin-B12-dependent methylmalonyl-CoA mutase lacks sufficient Cbl to convert L-methylmalonyl-CoA to succinyl-CoA.
Samples (n = 1092 from 301 individuals) from MARLINA-T2D (360 participants) at baseline (V3) and at end of the trial (V7: baseline [V3] + 6 months) were analyzed to determine pMMA and uMMA levels in a subset of the study cohort (Tables 2 and 3). Overall, no significant differences in MMA levels between placebo and linagliptin were observed, but significant differences were seen in different ethnic groups (Table 3). In individuals of Asian descent, significantly higher pMMA (p < 0.01, Cohen's d estimate 0.99 [95% CI 0.77-1.20]) and uMMA levels   Supplementary Fig. S2), indicating processing of CD320 by DPP-4.
To verify the hypothesis of increased CD320 in organs due to linagliptin treatment, we measured CD320 levels using a commercially available ELISA and evaluated the use of antibodies for capturing CD320 from tissue extracts. Surprisingly, CD320 protein could not be detected. Next, we evaluated two antibodies (AB1 and AB2) to capture the CD320 protein. AB1 was not able to capture the protein whereas AB2 recognized the HIS-tag of the expressed protein only ( Supplementary Fig. S3). Although processing was observed in vitro, ultimately, we were unable to determine CD320 levels and its processing in the tissue samples.

Discussion
Cbl subsumes several chemical forms of vitamin B12 and is classified by its axial ligand of the cobalt ion. Dietary B12 is often taken up as CNCbl or OHCbl and is converted by humans and animals into active Cbl compounds involving removal of the upper-axial ligand yielding the one-electron reduced intermediate cob(II)alamin for Cbl dependent enzymes. The dietary uptake, transport, and cellular influx of Cbl requires a complex process involving multiple proteins and receptors ( Supplementary Fig. S4) 7 . In an animal study (Exp#1 , Table 1), a significant accumulation of CNCbl in murine kidney (20-fold), liver (twofold) and heart (twofold) following linagliptin treatment over 12 weeks combined with a normal Cbl diet was observed. Shortening the duration of linagliptin treatment to 4 days and increasing the dietary amount of Cbl in a subsequent animal experiment (Exp#3 , Table 1) attenuated the effect of linagliptin on Cbl accumulation, whereas in animals with a normal Cbl diet (Exp#4), an approximate 20% increase of intracellular Cbl levels (spleen and heart) following short-term (2 weeks) linagliptin treatment was seen. It is worth noting that all animals were normoglycemic apart from the animals in Exp#4.
To determine if the altered Cbl homeostasis with linagliptin seen in mice is also present in humans, MMA levels in samples from MARLINA-T2D were determined. We used MMA as a surrogate marker for Cbl homeostasis, as homocysteine, another marker used to determine Cbl homeostasis, could not be measured in plasma samples due its pre-analytical requirements for blood sampling. MMA is a sensitive but not an entirely specific marker. MMA levels may be influenced by renal function, age and a variety of other factors (e.g., microbiota).
Because, both high as well as low Cbl-diet abrogated the effect of linagliptin on tissue Cbl accumulation in mice, MMA in samples from individuals in the MARLINA-T2D clinical trial were grouped into low, medium and high levels. In line with our animal data, analysis of the MARLINA-T2D data suggests that linagliptin can affect medium MMA levels: Placebo-treated individuals possess significantly higher MMA values in plasma  Tables 5 and 6, respectively. MMA, methylmalonic acid; n.s., not significant; V3, baseline; V7, end of study. www.nature.com/scientificreports/ and urine at V7 compared with linagliptin-treated individuals at V7, but not at V3. The Cohens' d effect size is greater for uMMA than for pMMA, which might be related to better diagnostic sensitivity of uMMA in detecting Cbl deficiency in patients with diabetes 8 . It is important to note that these effects observed in patients from MARLINA-T2D are modest; however, given that the observation period (six months) is relatively short, it is possible that incremental beneficial effect of linagliptin may be apparent over a longer treatment period. Furthermore, the clinical study population of interest was limited due to selection of appropriate samples based on estimated Cbl supply. We hypothesized that the effects of linagliptin on Cbl homeostasis in man and mice result from inhibition of DPP-4 dependent processing of major proteins involved in the Cbl pathway (Cubilin, Protein aminoless, Intrinsic factor, Haptocorrin, Megalin, Transcobalamin-2, MRP1 and CD320). The N-terminus of the CD320 protein contains a prototypical DPP-4 cleavage motif 9 both in humans (S36P37) and mice (A29P30). All other major proteins of the Cbl pathway are devoid of this motif. CD320‚ is a cell-surface receptor for transcobalamin saturated with cobalamin (TCbl) and plays an important role in cobalamin uptake. Loss of function mutations result in functional B12 deficiency [10][11][12] . To determine if CD320 is a potential substrate for DPP-4, we performed  www.nature.com/scientificreports/ in-vitro cleavage experiments of CD320. Incubation of CD320 in the presence of DPP-4 significantly increased the N-terminal dipeptide truncation and was inhibited by linagliptin, which strongly suggests that CD320 is indeed a substrate of DPP-4. DPP-4 is expressed on the plasma membrane but also exists in a soluble form 13 . It is conceivable that DPP-4 is capable of processing CD320 on the plasma membrane. A previous study 4 demonstrated the activity of DPP-4, its inhibition by linagliptin treatment in kidney tissue homogenates and the N-Terminal processing of proteins/peptides in tissue samples.. Additionally, we have recently shown inhibition of in situ activity of DPP-4 in rat kidney by linagliptin 14 .
We aimed to determine CD320 in tissue samples by using a commercially available ELISA and antibody capturing. However, both approaches were unsuccessful due to analytical challenges (Supp. Fig. S3).
CD320 expression appears to be tightly regulated according to the proliferative and differentiation status of the cell. Of note, mRNA analyses (of heart and kidney tissues from Exp#1, Table 1) did not reveal any significant differences in CD320 expression between control and linagliptin-treated animals (data not shown). In a recently published paper 15 , the authors reported that the binding of TC-Cbl to CD320 initiates internalization of the receptor complex (routed to the endolysosomal network), CD320 and transcobalamin is then proteolytically degraded, and Cbl is finally exported into the cytosolic compartment. Therefore, the expression of receptors on the cell surface is dependent on the synthesis of new receptors. The authors induced internalization by incubating cells in medium with excess of TC-Cbl. This is in accordance with the observations we obtained i.e., adding significant amounts of Cbl to the diet resulted in non-significant differences in intracellular Cbl levels between controls and linagliptin-treated animals because of the supposedly decreased CD320 density 16 thus reducing the potential DPP-4 processing thereby mitigating any linagliptin effect.
In summary, we demonstrate that linagliptin increased organ levels of Cbl in mice and resulted in sustained constant medium MMA levels in humans and speculate that these effects of linagliptin result from inhibition of the proteolytic cleavage of CD320 via its N-terminal DPP-4 cleavage site. However, we failed to demonstrate the processing of CD320 in tissue samples and did perform a post-hoc MMA analysis in already collected samples impeding the analysis of homocysteine due to its pre-analytical requirements. Since we analyzed a limited set of organs, we cannot exclude the possibility of decreased uptake of Cbl in other organs (e.g., brain or bone marrow). Nevertheless, we did conduct four independent animal experiments using semi-quantitative mass spectrometry to determine CNCbl and OHCBl in 150 tissue samples and could translate the findings to humans using MMA in a moderate sample size (n > 160 subjects) in two different specimens (plasma and urine) at two points in time thus increasing confidence in the conclusion drawn. www.nature.com/scientificreports/

Material and methods
Ethics statement. All animal experiments were performed with permission of the local authorities and conducted in accordance with the German legislation and the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were conducted in accordance with the Practice Guidelines for Laboratory animals. The study was carried out in compliance of the ARRIVE guidelines. The experiment was carried out under supervision of an experienced and certified person at Boehringer Ingelheim, and the protocol was approved by the Boehringer Ingelheim animal welfare committee. The MARLINA-T2D protocol was approved by independent ethics committees and institutional review boards at each participating center, and the study was conducted according to the principles of the Declaration of Helsinki and the ICH Harmonised Tripartite Guidelines for Good Clinical Practice. All individuals provided written informed consent prior to participation.
Animals and study design. Experiment #1. Animals were maintained under normal chow with standard 30 µg/kg CNCbl concentration for three months. Linagliptin concentration was 83 mg linagliptin/kg chow. Animals have been 5/6 nephrectomized to induced renal failure. Kidney, heart and liver were analyzed after three months for VitB12 content. The study is described in detail in a previous publication 4 . Experiment #2. Male C57BL6/J mice (age 10-11 weeks) obtained from Janvier Labs (Le Genest-Saint-Isle, France) were fed a 150 µg CNCbl/kg diet (n = 5) supplemented with 83 mg/kg linagliptin (n = 5) for 4 weeks. Control animals (n = 5) during the same period were given a diet containing 50 µg CNCbl/kg chow without linagliptin. At the end of the study, the kidney was analyzed for Vit-B12 content.
Experiment#3. Male mice from Janvier (10-11 weeks) were fed 2 weeks and 4 days a diet containing 150 µg CNCbl/kg chow (control animals; n = 5); and linagliptin treated animal (n = 5) were fed the same diet of 150 µg CNCbl/kg for 2 weeks and subsequently treated with for 4 days with 3 mg/kg/d linagliptin. At the end of the study kidney, heart, and liver were analyzed for Vit-B12 content.
Experiment #4. Mice from Janvier arrived at 5 weeks of age and were made diet-induced obese (DIO) by offering ad libitum access to tap water and high-fat diet (5.15 kcal/g; 60 kcal-% fat, 20 kcal-% carbohydrate, 20% kcal-% protein) for 26-27 weeks before study start. The CNCbl content was 30 µg/kg chow. Mice (n = 4 per group) received either vehicle or linagliptin (3 mg/kg/d) for the following 14 days. Kidney, heart, and spleen were analyzed for Vit-B12 content. The study is described in detail recently 17 . Material from this study was taken in order to translate the impaired metabolic situation from patients also to mice. This method has been previously described elsewhere 18 . Briefly, low molecular weight entities were extracted from tissues, separated by reversed phase liquid chromatography and an aliquot of each lyophilized fraction was re-suspended in 15 µl of a matrix solution containing alpha-cyano-4-hydroxy cinnamic acid (HCCA, 8 mg/ ml, Fluka) with 6-desoxy-L-galactose additive in 50/49/1 (v/v/v) 0.2% TFA/acetonitrile/acetone. 0.5 μl of the sample/matrix mixture were spotted on a target and subjected to MALDI-TOF-MS. After MS data acquisition, spectra were analyzed 19 . To semi-quantify CNCbl and OHCbl, 8 fmol of angiotensin or bradykinin was added prior to MS-measurement and resulting mass spectra were standardized to the intensity of angiotensin (CN) or bardykinin (OH). Two different standards for standardization for each Cbl type were used because of the background in different chromatographic fractions.
Exp#2 served as a verification experiment to demonstrate that elevated Cbl levels can be detected in tissue samples using MS. Therefore, linagliptin treated animals were fed a higher amount of Cbl (150 µg/kg chow) in comparison to controls (50 µg /kg chow). The resulting data (Fig. 1, Table 1) shows a significant accumulation of CNCbl in the kidneys thus strongly suggesting that the MS assay is capable to relatively quantify Cbl.
For mass calibration in all measurements, a mixture of standard peptides in a mass range from 1 to 6 kDa were used. The calibration spots were homogeneously distributed over the target plate. All measurements were calibrated using a default calibration, which was updated directly prior to the sample analysis using the automatic plate calibration function of the Explorer software package (Applied Biosystems, V. 2.0). For MS/MS experiments, fragmentation was carried out in the collision-induced dissociation (CID) mode of the MALDI-TOF/ TOF mass spectrometer with collision energy of 1 keV and ambient air as the collision gas at a typical pressure of 4*10-7 torr. MS/MS spectra were subsequently noise filtered and peak de-isotoped. The chromatograms and exemplary MS-MS fragment spectra of the four Cbl variants are given in Supplemental Fig. S5. MMA assay. Human plasma from the MARLINA trial was collected via venous puncture in EDTA-coated tubes and plasma was separated by centrifugation (2000 × g for 10 min at 4 °C). Urine was centrifuged (2000 × g for 10 min at 4 °C) and aliquots were immediately stored at − 80 °C. The assays were conducted mid-2017 for urine and early 2018 for plasma. A competitive ELISA kit (Catalog No: E2091Ge, Wuhan Eiaab Science Co., Ltd., Guangguguoji, China) was used according to the instructions by the manufacturer to determine MMA levels in urine and plasma. Briefly, a dilution series prepared from the supplied MMA standard was used to calibrate the  20 , as direct determinations of the N-terminal truncated forms are challenging. Resulting digests were analyzed using reverse-phase chromatography and mass spectrometry. Peptides were identified using MS-MS fragmentation. In total, 14 independent incubations experiments (CD320: 6, CD320 + DPP-4: 6, CD320 + DPP-4 + linagliptin: 2) were performed and measured in MS in triplicate. The digestion of CD320 yields, among others, a tryptic peptide (SPLSTPTSAQAAGPSSGSCPPTK) representing the N-terminus of CD320 (CD320AA36-58). Prior incubation of CD320 with DPP-4 results in an increase of the truncated form (CD320AA38-58) of the said peptide i.e., a surrogate marker for DPP-4 cleavage. For analysis, the intensity-ratios between CD320AA38-58 and the sum of CD320AA36-58 and CD320AA38-58 were calculated to estimate the conversion rate.
Data analysis. For data analysis, R (A Language and Environment for Statistical Computing: www.r-proje ct. org) version 4.1.0, including packages tidyverse, rstatix, effsize, MALDIquant and reshape2 was used. For statistical testing Kruskal-Wallis rank sum test, Wilcoxon signed rank test with continuity correction, one-way ANOVA and Welch t-test were employed. For calculation of effect sizes, Cohen's d estimate was used. Confidence intervals were calculated by bootstrap. Kernel density estimates were calculated using ggplot2.

Data availability
To ensure independent interpretation of clinical study results and enable authors to fulfill their role and obligations under the ICMJE criteria, Boehringer Ingelheim grants all external authors access to relevant clinical study data pertinent to the development of the publication. In adherence with the Boehringer Ingelheim Policy on Transparency and Publication of Clinical Study Data, scientific and medical researchers can request access to clinical study data when it becomes available on https:// vivli. org/ and earliest after publication of the primary manuscript in a peer-reviewed journal, regulatory activities are complete and other criteria are met. Please visit https:// www. mystu dywin dow. com/ msw/ datas haring for further information. www.nature.com/scientificreports/